Mitogen activated c-Fos regulating kinase, FRK

ABSTRACT

An isolated nitrogen activated c-Fos regulating kinase polypeptide (FRK) having a molecular weight of about 88-kD as determined by reducing SDS-PAGE, having threonine and serine kinase activity, phosphorylating the c-Fos activation domain at amino acid residue Thr 232 and having polynucleotide sequences and a method of detection for FRK are provided herein. Also described are methods for identifying compositions which affect FRK activity, thereby affecting c-Fos activation and subsequent activation of genes associated with AP-1 sites.

This invention was made with Government support under Grant Nos. CA50528and ES-06376 awarded by the National Institutes of Health; Grant No.MG-202393 awarded by the American Cancer Society, Council for TobaccoResearch and Grant No. 3RT-0138 awarded by the Tobacco-Related DiseaseResearch Program. The Government has certain rights in this invention.

This is a divisional of copending application Ser. No. 08/315,067, filedSep. 29, 1994.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to the field of protein kinases,oncogenes and oncoproteins and, specifically, to a protein kinase whichphosphorylates and potentiates the activity of c-Fos.

2. Description of Related Art

A number of viral and cellular genes have been identified as potentialcancer genes, collectively referred to as oncogenes. The cellularhomologs of viral oncogenes, the proto-oncogenes or c-oncogenes, act inthe control of cell growth and differentiation or mediate intracellularsignaling systems. The products of oncogenes are classified according totheir cellular location, for example, secreted, surface, cytoplasmic,and nuclear oncoproteins.

Proto-oncogenes which express proteins which are targeted to the cellnucleus make up a small fraction of oncogenes. These nuclearproto-oncoproteins typically act directly as transactivators andregulators of RNA and DNA synthesis. Nuclear oncogene products have theability to induce alterations in gene regulation leading to abnormalcell growth and ultimately neoplasia. Examples of nuclear oncogenesinclude the myc, ski, myb, fos and jun genes.

The c-Fos protein, encoded by the c-fos proto-oncogene, is an importantcomponent of the dimeric, sequence specific, transcriptional activator,AP-1. Many proteins cooperate with each other in the activation oftranscription from specific promoters. Through this cooperation, a genecan be transcribed and a protein product generated. Members of the Fosproto-oncogene family, along with members of the Jun gene family, formstable complexes which bind to DNA at an AP-1 site. The AP-1 site islocated in the promoter region of a large number of genes. Binding ofthe Fos/Jun complex activates transcription of a gene associated with anAP-1 site. In cells that have lost their growth regulatory mechanisms,it is believed that this Fos/Jun complex may "sit" on the AP-1 site,causing overexpression of a particular gene. Since many proliferativedisorders result from the overexpression of an otherwise normal gene,such as a proto-oncogene, it would be desirable to identify compositionswhich interfere with the excessive activation of these genes.

Ras proteins exert their mitogenic and oncogenic effects by activatingprotein kinase cascades leading to phosphorylation of nucleartranscription factors. AP-1, as described above, is a heterodimericcomplex of Jun and Fos proteins, which activates mitogen-inducible genesand is also a major nuclear target of Ras. Ras can stimulate AP-1activity through c-fos induction (Angel and Karin, Biophys. Acta.,1072:129-157,1991: Herrlich and Ponta, Trends Genet., 5:112-116, 1989),a process likely to be mediated by the ERK1 and ERK2 mitogen activatedprotein (MAP) kinases (G. Thomas, Cell, 68:3-6, 1992), throughphosphorylation of Elk-1/TCF (Sharrocks, and Shaw, Nature, 358:414-417,1992; Wynne, et al., Cell, 73:381-393, 1993; Zinck, et al., EMBO J.,12:2377-2387, 1993). However, besides inducing fos and jun genetranscription, mitogens and Ras proteins enhance AP-1 activity throughphosphorylation of c-Jun (Binetruy, et al., Nature, 351:122-127, 1991;Smeal, et al., Nature, 354:494-496, 1991; Pulverer, et al., Nature,353:683-689, 1991). Through an autoregulatory loop, phosphorylation ofthe c-Jun activation domain leads to c-jun induction (Angel and Karin,supra). Recently, Ras- and UV-responsive protein kinases thatphosphorylate c-Jun on serines (Ser) 63 and 73 and stimulate itstranscriptional activity were identified (Hibi, et al., Genes & Dev.,7:2135-2148, 1993).

These proline-directed kinases, termed JNK, for c-Jun-N-terminal kinase,are novel MAP kinases (Derijard, et al., Cell, 75:1025-1037, 1994). Itis not clear, however, whether c-Jun is the only recipient and whetherJNK is the only transducer of the Ras signal to AP-1 proteins. A shortsequence surrounding the major JNK phosphorylation site of c-Jun (Ser73)is conserved in c-Fos and is part of its activation domain (Sutherland,et al., Genes & Dev., 6:1810-1819, 1992). The present inventiondemonstrates that Ras does indeed augment c-Fos transcriptional activitythrough phosphorylation at Thr232, the homolog of Ser73 of c-Jun.However, this is mediated by a novel Ras- and mitogen-responsiveproline-directed protein kinase that is different from JNKs and ERKs.

For many years, various drugs have been tested for their ability toalter the expression of genes or the translation of their messages intoprotein products. One problem with existing drug therapy is that ittends to act indiscriminately and affects healthy cells as well asneoplastic cells. This is a major problem with many forms ofchemotherapy where there are severe side effects primarily due to theaction of toxic drugs on healthy cells.

In view of the foregoing, there is a need to identify specific targetsin the abnormal cell which are associated with the overexpression ofgenes whose expression products are implicated in cell proliferativedisorders, in order to decrease potential negative effects on healthycells. The present invention provides such a target.

SUMMARY OF THE INVENTION

The present invention provides a novel Fos regulating protein kinase(FRK) which phosphorylates c-Fos and potentiates its activity. FRK ischaracterized by having a molecular weight of about 88 kD (as determinedby reducing SDS-polyacrylamide gel electrophoresis (PAGE)) and havingthreonine and serine kinase activity. Specifically, FRK phosphorylatesthreonine residue 232 of c-Fos.

Since the product of the fos proto-oncogene is a transactivator proteinwhich binds at AP-1 sites, regulation of c-Fos activation may beimportant in affecting normal gene expression and growth control in acell. The discovery of FRK provides a means for identifying compositionswhich affect FRK activity, thereby affecting c-Fos activation andsubsequent activation of genes associated with AP-1 sites.

The identification of FRK now allows the detection of the level ofspecific kinase activity associated with activation of c-Fos and AP-1.In addition, the invention provides a method of treating a cellproliferative disorder associated with FRK by administering to a subjectwith the disorder, a therapeutically effective amount of a reagent whichmodulates FRK activity.

The invention also provides a synthetic peptide comprising the FRKbinding region on c-Fos which corresponds to amino acids 226-236 (₂₂₆GLPEASTPES-E₂₃₆ (SEQ ID NO:1)). The peptide is useful as a competitiveinhibitor of the naturally occurring c-Fos in situations where it isdesirable to decrease the amount of c-Fos activation by FRK.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B show diagrams illustrating the salient features of c-Fosand the GHF1-cFos (210-313) chimera, including the position of the HOB1and HOB2 regions and a sequence alignment of the HOB1 regions of c-Junand c-Fos.

FIG. 1C shows Ras-, UV-, or TPA- induced activity of c-Fos.

FIG. 2 shows transactivation of c-Fos constructs in the presence orabsence of Ha-Ras.

FIGS. 3A-C show 2-D phosphopeptide analysis after stimulation of c-Fosphosphorylation on Thr232 by co-expression of activated Ha-Ras.

FIG. 4A shows SDS-PAGE of c-Fos immunoprecipitates after in vivophosphorylation of c-Fos. (C=control; T=TPA (100 ng/ml); U=UVirradiation; R=cotransfection with pSG-Ha-Ras-Leu61).

FIG. 4B shows SDS-PAGE of phosphorylation of c-Fos and c-Jun aftertreatment with JNK1 or ERK1/2. (W=wild-type c-Fos; M=c-fos (T232A)).

FIG. 4C shows a 2-D tryptic peptide map of wild-type c-Fosphosphorylated in vitro by ERK1/2 and 1:1 mixture between c-Fosphosphorylated in vitro by ERK1/2 and c-Fos labeled in vivo and isolatedfrom Ha-Ras cotransfected cells.

FIGS. 5A and 5B show Luc reporter activity indicating c-Fostranscriptional activity after treatment with or without EGF.

FIG. 6A shows an in-gel kinase assay identifying the mitogen-activatedkinase phosphorylating c-Fos at Thr232. The 88 kD band corresponding toFRK is indicated.

FIG. 6B shows whole cell extracts analyzed by an in-gel kinase assayafter treatment of cells with EGF (E), EGF and dexamethasone (D and E)or untreated (C) PC12 Ha-Ras(Asn17)!.

FIG. 6C shows phosphopeptide mapping of c-Fos and c-Fos (T232A)phosphorylated by FRK.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel protein kinase (FRK) which bindsto a well-defined region of the c-Fos proto-oncoprotein andphosphorylates a site within its activation domain. The phosphorylationof this site increases the ability of c-Fos to stimulate transcriptionand mediate oncogenic transformation.

The activity of c-Fos is regulated by phosphorylation. Various stimuli,including transforming oncogenes and mitogens, induce thephosphorylation of threonine 232 in c-Fos's activation domain, therebypotentiating its transactivation function. The invention relates to anisolated polypeptide characterized by having a molecular weight of 88 kDas determined by reducing SDS-PAGE, having threonine and serine kinaseactivity, and capable of phosphorylating the c-Fos activation domain.

The term "isolated" means any FRK polypeptide of the present invention,or any gene encoding a FRK polypeptide, which is essentially free ofother polypeptides or genes, respectively, or of other contaminants withwhich the FRK polypeptide or gene might normally be found in nature.

The invention includes a functional polypeptide, FRK, and functionalfragments thereof. As used herein, the term "functional polypeptide"refers to a polypeptide which possesses a biological function oractivity which is identified through a defined functional assay andwhich is associated with a particular biologic, morphologic, orphenotypic alteration in the cell. The biological function, for example,can vary from a polypeptide fragment as small as an epitope to which anantibody molecule can bind to a large polypeptide which is capable ofparticipating in the characteristic induction or programming ofphenotypic changes within a cell. An enzymatically functionalpolypeptide or fragment of FRK possesses c-Fos activation domain kinaseactivity, therefore, an in-gel kinase assay, for example, can beperformed to identify a functional polypeptide. A "functionalpolynucleotide" denotes a polynucleotide which encodes a functionalpolypeptide as described herein.

Minor modifications of the FRK primary amino acid sequence may result inproteins which have substantially equivalent activity as compared to theFRK polypeptide described herein. Such modifications may be deliberate,as by site-directed mutagenesis, or may be spontaneous. All of thepolypeptides produced by these modifications are included herein as longas the kinase activity of FRK is present. Further, deletion of one ormore amino acids can also result in a modification of the structure ofthe resultant molecule without significantly altering its kinaseactivity. This can lead to the development of a smaller active moleculewhich would have broader utility. For example, it is possible to removeamino or carboxy terminal amino acids which may not be required for FRKkinase activity.

The FRK polypeptide of the invention also includes conservativevariations of the polypeptide sequence. The term "conservativevariation" as used herein denotes the replacement of an amino acidresidue by another, biologically similar residue. Examples ofconservative variations include the substitution of one hydrophobicresidue such as isoleucine, valine, leucine or methionine for another,or the substitution of one polar residue for another, such as thesubstitution of arginine for lysine, glutamic for aspartic acids, orglutamine for asparagine, and the like. The term "conservativevariation" also includes the use of a substituted amino acid in place ofan unsubstituted parent amino acid provided that antibodies raised tothe substituted polypeptide also immunoreact with the unsubstitutedpolypeptide.

The invention also provides a synthetic peptide which binds to the c-Foskinase, FRK. The amino acid sequence of SEQ ID NO:1, and conservativevariations, comprises the synthetic peptide of the invention. Thissequence represents amino acids of 226-236 of c-Fos polypeptide (FIG. 1;Sutherland, J. A., et al., Genes & Dev., 6:1810, 1992) and includesThr232 which is phosphorylated by FRK. As used herein, the term"synthetic peptide" denotes a peptide which does not comprise an entirenaturally occurring protein molecule. The peptide is "synthetic" in thatit may be produced by human intervention using such techniques aschemical synthesis, recombinant genetic techniques, or fragmentation ofwhole antigen or the like.

Peptides of the invention can be synthesized by such commonly usedmethods as t-BOC or FMOC protection of alpha-amino groups. Both methodsinvolve stepwise syntheses whereby a single amino acid is added at eachstep starting from the C terminus of the peptide (See, Coligan, et al.,Current Protocols in Immunology, Wiley Interscience, 1991, Unit 9).Peptides of the invention can also be synthesized by the well knownsolid phase peptide synthesis methods described by Merrifield (J. Am.Chem. Soc., 85:2149, 1962), and Stewart and Young, Solid Phase, PeptidesSynthesis, (Freeman, San Francisco, 1969, pp. 27-62), using acopoly(styrene-divinylbenzene) containing 0.1-1.0 mMol amines/g polymer.On completion of chemical synthesis, the peptides can be deprotected andcleaved from the polymer by treatment with liquid HF-10% anisole forabout 1/4-1 hours at 0° C. After evaporation of the reagents, thepeptides are extracted from the polymer with 1% acetic acid solutionwhich is then lyophilized to yield the crude material. This can normallybe purified by such techniques as gel filtration on Sephadex G-15 using5% acetic acid as a solvent. Lyophilization of appropriate fractions ofthe column will yield the homogeneous peptide or peptide derivatives,which can then be characterized by such standard techniques as aminoacid analysis, thin layer chromatography, high performance liquidchromatography, ultraviolet absorption spectroscopy, molar rotation,solubility, and quantitated by the solid phase Edman degradation.

The invention also provides polynucleotides which encode the FRKpolypeptide of the invention and the synthetic peptide of SEQ ID NO:1.As used herein, "polynucleotide" refers to a polymer ofdeoxyribonucleotides or ribonucleotides, in the form of a separatefragment or as a component of a larger construct. DNA encoding thepolypeptide of the invention can be assembled from cDNA fragments orfrom oligonucleotides which provide a synthetic gene which is capable ofbeing expressed in a recombinant transcriptional unit. Polynucleotidesequences of the invention include DNA, RNA and cDNA sequences.

DNA sequences of the invention can be obtained by several methods. Forexample, the DNA can be isolated using hybridization procedures whichare well known in the art. These include, but are not limited to:(1)hybridization of probes to genomic or CDNA libraries to detect sharednucleotide sequences; (2) antibody screening of expression libraries todetect shared structural features and (3) synthesis by the polymerasechain reaction (PCR).

Hybridization procedures are useful for the screening of recombinantclones by using labeled mixed synthetic oligonucleotide probes whereeach probe is potentially the complete complement of a specific DNAsequence in the hybridization sample which includes a heterogeneousmixture of denatured double-stranded DNA. For such screening,hybridization is preferably performed on either single-stranded DNA ordenatured double-stranded DNA. Hybridization is particularly useful inthe detection of cDNA clones derived from sources where an extremely lowamount of mRNA sequences relating to the polypeptide of interest arepresent. In other words, by using stringent hybridization conditionsdirected to avoid non-specific binding, it is possible, for example, toallow the autoradiographic visualization of a specific cDNA clone by thehybridization of the target DNA to that single probe in the mixturewhich is its complete complement (Wallace, et al., Nucleic AcidResearch, 9:879, 1981).

The development of specific DNA sequences encoding FRK can also beobtained by: (1) isolation of double-stranded DNA sequences from thegenomic DNA; (2) chemical manufacture of a DNA sequence to provide thenecessary codons for the polypeptide of interest; and (3) in vitrosynthesis of a double-stranded DNA sequence by reverse transcription ofmRNA isolated from a eukaryotic donor cell. In the latter case, adouble-stranded DNA complement of mRNA is eventually formed which isgenerally referred to as cDNA. Of these three methods for developingspecific DNA sequences for use in recombinant procedures, the isolationof genomic DNA isolates is the least common. This is especially truewhen it is desirable to obtain the microbial expression of mammalianpolypeptides due to the presence of introns.

The synthesis of DNA sequences is frequently the method of choice whenthe entire sequence of amino acid residues of the desired polypeptideproduct is known. When the entire sequence of amino acid residues of thedesired polypeptide is not known, the direct synthesis of DNA sequencesis not possible and the method of choice is the synthesis of cDNAsequences. Among the standard procedures for isolating cDNA sequences ofinterest is the formation of plasmid- or phage-carrying cDNA librarieswhich are derived from reverse transcription of mRNA which is abundantin donor cells that have a high level of genetic expression. When usedin combination with polymerase chain reaction technology, even rareexpression products can be cloned. In those cases where significantportions of the amino acid sequence of the polypeptide are known, theproduction of labeled single or double-stranded DNA or RNA probesequences duplicating a sequence putatively present in the target cDNAmay be employed in DNA/DNA hybridization procedures which are carriedout on cloned copies of the cDNA which have been denatured into asingle-stranded form (Jay et al., Nucl. Acid Res., 11:2325, 1983).

A cDNA expression library, such as lambda gt11, can be screenedindirectly for FRK polypeptide having at least one epitope, usingantibodies specific for FRK. Such antibodies can be either polyclonallyor monoclonally derived and used to detect expression product indicativeof the presence of FRK cDNA. Polynucleotide sequences encoding apolypeptide having an amino acid sequence possessing at least oneepitope to which an antibody to FRK binds, are included in theinvention.

A polynucleotide sequence can be deduced from the genetic code, however,the degeneracy of the code must be taken into account. Polynucleotidesof the invention include sequences which are degenerate as a result ofthe genetic code. The polynucleotides of the invention include sequencesthat are degenerate as a result of the genetic code. There are 20natural amino acids, most of which are specified by more than one codon.Therefore, as long as the amino acid sequence of FRK results in afunctional (e.g., retains kinase activity) polypeptide (at least, in thecase of the sense polynucleotide strand), all degenerate nucleotidesequences are included in the invention.

The polynucleotide encoding FRK includes the full-length nucleotidesequence, as well as nucleic acid sequences complementary to thatsequence. A complementary sequence may include an antisense nucleotide.When the sequence is RNA, the deoxynucleotides A, G, C, and T arereplaced by ribonucleotides A, G, C, and U, respectively. Also includedin the invention are fragments of the above-described nucleic acidsequences that are at least 15 bases in length, which is sufficient topermit the fragment to selectively hybridize to DNA that encodes theprotein under physiological conditions.

Since a polynucleotide sequence of the invention encodes essentially theentire FRK molecule it is now a matter of routine to prepare, subclone,and express smaller polypeptide fragments of polynucleotide from this ora corresponding polynucleotide sequence which would encode as few as oneepitope for antibodies to FRK. The presence of such an epitope on acloned polypeptide can then be confirmed using, for example, an antibodywhich binds to FRK.

The polynucleotide sequence for FRK also includes sequencescomplementary to the polynucleotide encoding FRK (antisense sequences).Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule (Weintraub, ScientificAmedcan, 262:40, 1990). The invention embraces all antisensepolynucleotides capable of inhibiting production of FRK polypeptide. Inthe cell, the antisense nucleic acids hybridize to the correspondingmRNA, forming a double-stranded molecule. The antisense nucleic acidsinterfere with the translation of the mRNA since the cell will nottranslate a mRNA that is double-stranded. Antisense oligomers of about15 nucleotides are preferred, since they are easily synthesized and areless likely to cause problems than larger molecules when introduced intothe target FRK-producing cell. The use of antisense methods to inhibitthe translation of genes is well known in the art (Marcus-Sakura, Anal.Biochem., 172:289, 1988).

In addition, ribozyme nucleotide sequences for FRK are included in theinvention. Ribozymes are RNA molecules possessing the ability tospecifically cleave other single-stranded RNA in a manner analogous toDNA restriction endonucleases. Through the modification of nucleotidesequences which encode these RNAS, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, J. Amer. Med.-Assn., 260:3030, 1988). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff, Nature, 334:585, 1988) and "hammerhead"-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while "hammerhead"-type ribozymes recognize base sequences 11-18bases in length. The longer the recognition sequence, the greater thelikelihood that that sequence will occur exclusively in the target mRNAspecies. Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating a specific mRNA species and18-based recognition sequences are preferable to shorter recognitionsequences.

The FRK polypeptides of the invention can also be used to produceantibodies which are immunoreactive or bind to epitopes of the FRKpolypeptides. Antibodies of the invention also include antibodies whichbind to the synthetic peptide in SEQ ID NO:1. Antibody which consistsessentially of pooled monoclonal antibodies with different epitopicspecificities, as well as distinct monoclonal antibody preparations areprovided. Monoclonal antibodies are made from antigen containingfragments of the protein by methods well known in the art (Kohler, etal., Nature, 256:495, 1975; Current Protocols in Molecular Biology,Ausubel, et al., ed., 1989).

The term "monoclonal antibody" refers to a population of one species ofantibody molecule of determined (known) antigen-specificty. A monoclonalantibody contains only one species of antibody combining site capable ofimmunoreacting with a particular antigen and thus typically displays asingle binding affinity for that antigen. A monoclonal antibody maytherefore contain a bispecific antibody molecule having two antibodycombining sites, each immunospecific for a different antigen.

The term "antibody" as used in this invention includes intact moleculesas well as fragments thereof, such as Fab, F(ab')₂, and Fv which arecapable of binding the epitopic determinant. These antibody fragmentsretain some ability to selectively bind with its antigen or receptor andare defined as follows:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab', the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab' fragmentsare obtained per antibody molecule;

(3) (Fab')₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab')₂ is a dimer of two Fab' fragments held together by twodisulfide bonds;

(4) Fv, defined as a genetically engineered fragment containing thevariable region of the light chain and the variable region of the heavychain expressed as two chains; and

(5) Single chain antibody ("SCA"), defined as a genetically engineeredmolecule containing the variable region of the light chain, the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule.

Methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1988), incorporated herein by reference).

As used in this invention, the term "epitope" means any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

Antibodies which bind to the FRK polypeptide of the invention can beprepared using an intact polypeptide or fragments containing smallpeptides of interest as the immunizing antigen. The polypeptide or apeptide such as SEQ ID NO:1 used to immunize an animal can be derivedfrom translated cDNA or chemical synthesis which can be conjugated to acarrier protein, if desired. Such commonly used carriers which arechemically coupled to the peptide include keyhole limpet hemocyanin(KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.The coupled peptide is then used to immunize the animal (e.g., a mouse,a rat, or a rabbit).

If desired, polyclonal or monoclonal antibodies can be further purified,for example, by binding to and elution from a matrix to which thepolypeptide or a peptide to which the antibodies were raised is bound.Those of skill in the art will know of various techniques common in theimmunology arts for purification and/or concentration of polyclonalantibodies, as well as monoclonal antibodies (See for example, Coligan,et al., Unit 9, Current Protocols in Immunology, Wiley Interscience,1991, incorporated by reference).

It is also possible to use the anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the"image" of the epitope bound by the first monoclonal antibody. Thus, inthe present invention, an anti-idiotype antibody produced from anantibody which binds to the synthetic peptide of the invention can bindto the site on FRK which binds to c-Fos, thereby preventing FRK frombinding to and phosphorylating c-Fos.

Polynucleotide sequences encoding the polypeptide or synthetic peptide(SEQ ID NO:1) of the invention can be expressed in either prokaryotes oreukaryotes. Hosts can include microbial, yeast, insect and mammalianorganisms. Methods of expressing DNA sequences having eukaryotic orviral sequences in prokaryotes are well known in the art. Biologicallyfunctional viral and plasmid DNA vectors capable of expression andreplication in a host are known in the art. Such vectors are used toincorporate DNA sequences of the invention.

DNA sequences encoding the polypeptides can be expressed in vitro by DNAtransfer into a suitable host cell. "Host cells" are cells in which avector can be propagated and its DNA expressed. The term also includesany progeny of the subject host cell. It is understood that all progenymay not be identical to the parental cell since there may be mutationsthat occur during replication. However, such progeny are included whenthe term "host cell" is used. Methods of stable transfer, in other wordswhen the foreign DNA is continuously maintained in the host, are knownin the art.

In the present invention, the FRK polynucleotide sequences may beinserted into a recombinant expression vector. The term "recombinantexpression vector" refers to a plasmid, virus or other vehicle known inthe art that has been manipulated by insertion or incorporation of thegenetic sequences. Such expression vectors contain a promoter sequencewhich facilitates the efficient transcription of the inserted geneticsequence of the host. The expression vector typically contains an originof replication, a promoter, as well as specific genes which allowphenotypic selection of the transformed cells. Vectors suitable for usein the present invention include, but are not limited to the T7-basedexpression vector for expression in bacteria (Rosenberg et al., Gene56:125, 1987), the pMSXND expression vector for expression in mammaliancells (Lee and Nathans, J. Biol. Chem. 263:3521, 1988) andbaculovirus-derived vectors for expression in insect cells. The DNAsegment can be present in the vector operably linked to regulatoryelements, for example, a promoter (e.g., T7, metallothionein I, orpolyhedrin promoters).

The vector may include a phenotypically selectable marker to identifyhost cells which contain the expression vector. Examples of markerstypically used in prokaryotic expression vectors include antibioticresistance genes for ampicillin (β-lactamases), tetracycline andchloramphenicol (chloramphenicol acetyl-transferase). Examples of suchmarkers typically used in mammalian expression vectors include the genefor adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo,G418), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase(HPH), thymidine kinase (TK), and xanthine guaninephosphoribosyltransferse (XGPRT, gpt).

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques which are well known to those skilled in theart. Where the host is prokaryotic, such as E. coli, competent cellswhich are capable of DNA uptake can be prepared from cells harvestedafter exponential growth phase and subsequently treated by the CaCl₂method by procedures well known in the art. Alternatively, MgCl₂ or RbClcan be used. Transformation can also be performed after forming aprotoplast of the host cell or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with DNA sequences encoding the polypeptides of theinvention, and a second foreign DNA molecule encoding a selectablephenotype, such as the herpes simplex thymidine kinase gene. Anothermethod is to use a eukaryotic viral vector, such as simian virus 40(SV40) or bovine papilloma virus, to transiently infect or transformeukaryotic cells and express the protein. (Eukaryotic Viral Vectors,Cold Spring Harbor Laboratory, Gluzman ed., 1982). Examples of mammalianhost cells include COS, BHK, 293, and CHO cells.

Isolation and purification of host cell expressed polypeptide, orfragments thereof, provided by the invention, may be carried out byconventional means including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.

The FRK protein kinase of the invention is useful in a screening methodfor identifying compounds or compositions which affect the activity ofthe kinase. Thus, in another embodiment, the invention provides a methodfor identifying a composition which affects a c-Fos kinase comprisingincubating the components, which include the composition to be testedand the kinase or a polynucleotide encoding the kinase, under conditionssufficient to allow the components to interact, then subsequentlymeasuring the effect the composition has on kinase activity or on thepolynucleotide encoding the kinase. The observed effect on the kinasemay be either inhibitory or stimulatory. For example, the increase ordecrease of kinase activity can be measured by adding a radioactivecompound to the mixture of components, such as ³² P-ATP, and observingradioactive incorporation into c-Fos or other suitable substrate forFRK, such as a peptide comprising SEQ ID NO:1, to determine whether thecompound inhibits or stimulates protein kinase activity. Apolynucleotide encoding the kinase may be inserted into an expressionvector and the effect of a composition on transcription of the kinasecan be measured, for example, by Northern blot analysis.

In another embodiment, the invention provides a method of treating acell proliferative disorder associated with FRK comprising administeringto a subject with the disorder a therapeutically effective amount ofreagent which modulates kinase activity. The term "therapeuticallyeffective" means that the amount of peptide, monoclonal antibody orantisense nucleotide, for example, which is used, is of sufficientquantity to ameliorate the FRK associated disorder. The term"cell-proliferative disorder" denotes malignant as well as non-malignantcell populations which morphologically often appear to differ from thesurrounding tissue. For example, the method may be useful in treatingmalignancies of the various organ systems, such as lung, breast,lymphoid, gastrointestinal, and genito-urinary tract as well asadenocarcinomas which include malignancies such as most colon cancers,renal-cell carcinoma, prostate cancer, non-small cell carcinoma of thelung, cancer of the small intestine and cancer of the esophagus.

The method is also useful in treating non-malignant orimmunological-related cell-proliferative diseases such as psoriasis,pemphigus vulgaris, Behcet's syndrome, acute respiratory distresssyndrome (ARDS), ischemic heart disease, post-dialysis syndrome,leukemia, rheumatoid arthritis, acquired immune deficiency syndrome,vasculitis, septic shock and other types of acute inflammation, andlipid histiocytosis. Essentially, any disorder which is etiologicallylinked to FRK kinase activity would be considered susceptible totreatment.

Treatment includes administration of a reagent which modulates FRKkinase activity. The term "modulate" envisions the suppression ofexpression of FRK when it is over-expressed, or augmentation of FRKexpression when it is underexpressed. It also envisions suppression ofphosphorylation of c-Fos, for example, by using the peptide of SEQ IDNO:1 as a competitive inhibitor of the natural c-Fos binding site in acell. When a cell proliferative disorder is associated with FRKoverexpression, such suppressive reagents as antisense FRKpolynucleotide sequence or FRK binding antibody can be introduced to acell. In addition, an anti-idiotype antibody which binds to a monoclonalantibody which binds a peptide of the invention may also be used in thetherapeutic method of the invention. Alternatively, when a cellproliferative disorder is associated with underexpression or expressionof a mutant FRK polypeptide, a sense polynucleotide sequence (the DNAcoding strand) or FRK polypeptide can be introduced into the cell.

The antibodies of the invention can be administered parenterally byinjection or by gradual infusion over time. The monoclonal antibodies ofthe invention can be administered intravenously, intraperitoneally,intramuscularly, subcutaneously, intracavity, or transdermally.

Preparations for parenteral administration of a peptide or an antibodyof the invention include sterile aqueous or non-aqueous solutions,suspensions, and emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media. Parenteral vehicles include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's, or fixed oils. Intravenous vehicles include fluid andnutrient replenishers, electrolyte replenishers (such as those based onRinger's dextrose), and the like. Preservatives and other additives mayalso be present such as, for example, antimicrobials, antioxidants,chelating agents, and inert gases and the like.

Polynucleotide sequences, including antisense sequences, can betherapeutically administered by various techniques known to those ofskill in the art. Such therapy would achieve its therapeutic effect byintroduction of the FRK polynucleotide, into cells of animals having theproliferative disorder. Delivery of FRK polynucleotide can be achievedusing free polynucleotide or a recombinant expression vector such as achimeric virus or a colloidal dispersion system. Especially preferredfor therapeutic delivery of nucleotide sequences is the use of targetedliposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. By inserting a FRK sequence into the viralvector, along with another gene which encodes the ligand for a receptoron a specific target cell, for example, the vector is now targetspecific. Retroviral vectors can be made target specific by inserting,for example, a polynucleotide encoding a sugar, a glycolipid, or aprotein. Preferred targeting is accomplished by using an antibody totarget the retroviral vector. Those of skill in the art will know of, orcan readily ascertain without undue experimentation, specificpolynucleotide sequences which can be inserted into the retroviralgenome to allow target specific delivery of the retroviral vectorcontaining the FRK polynucleotide.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence which enables the packaging mechanism to recognizean RNA transcript for encapsitation. Helper cell lines which havedeletions of the packaging signal include but are not limited to Ψ2,PA317 and PA12, for example. These cell lines produce empty virions,since no genome is packaged. If a retroviral vector is introduced intosuch cells in which the packaging signal is intact, but the structuralgenes are replaced by other genes of interest, the vector can bepackaged and vector virion produced. The vector virions produced by thismethod can then be used to infect a tissue cell line, such as NIH 3T3cells, to produce large quantities of chimeric retroviral virions.

Another targeted delivery system for FRK polynucleotides is a colloidaldispersion system. Colloidal dispersion systems include macromoleculecomplexes, nanocapsules, microspheres, beads, and lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles, andliposomes. The preferred colloidal system of this invention is aliposome. Liposomes are artificial membrane vesicles which are useful asdelivery vehicles in vitro and in vivo. It has been shown that largeunilamellar vesicles (LUV), which range in size from 0.2-4.0 um canencapsulate a substantial percentage of an aqueous buffer containinglarge macromolecules. RNA, DNA and intact virions can be encapsulatedwithin the aqueous interior and be delivered to cells in a biologicallyactive form (Fraley, et al., Trends Biochem. Sci., 6:77, 1981). Inaddition to mammalian cells, liposomes have been used for delivery ofpolynucleotides in plant, yeast and bacterial cells. In order for aliposome to be an efficient gene transfer vehicle, the followingcharacteristics should be present: (1) encapsulation of the genes ofinterest at high efficiency while not compromising their biologicalactivity; (2) preferential and substantial binding to a target cell incomparison to non-target cells; (3) delivery of the aqueous contents ofthe vesicle to the target cell cytoplasm at high efficiency; and (4)accurate and effective expression of genetic information (Mannino, etal, Biotechniques, 6:682, 1988).

The targeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The invention also provides a method for detecting a cell with FRKkinase activity or a cell proliferative disorder associated with FRKcomprising contacting a cell component with c-Fos kinase activity with areagent which binds to the component and measuring the interaction ofthe reagent with the component. Such reagents can be used to measurerelative levels of FRK expression compared to normal tissue. The cellcomponent can be nucleic acid, such as DNA or RNA, or protein. When thecomponent is nucleic acid, the reagent is a nucleic acid probe or PCRprimer. The interaction of a nucleic acid reagent with a nucleic acidencoding a polypeptide with c-Fos kinase activity is typically measuredusing radioactive labels, however, other types of labels will be knownto those of skill in the art. When the cell component is protein, thereagent is typically an antibody probe. The probes are directly orindirectly detectably labeled, for example, with a radioisotope, afluorescent compound, a bioluminescent compound, a chemiluminescentcompound, a metal chelator or an enzyme. Those of ordinary skill in theart will know of other suitable labels for binding to the antibody, orwill be able to ascertain such, using routine experimentation.

Preferably the probe for identification of a cell with FRK kinaseactivity is a c-Fos protein. FRK activity within a cell is measured bythe amount of phosphorylation of the c-Fos protein probe. For example,the amount of FRK activity in a cell extract can be measured by mixingthe extract with c-Fos protein or a peptide comprising SEQ ID NO:1, andadding a radioactive compound such as ³² P-ATP to the mixture ofcomponents. The amount of radioactivity that is incorporated into thec-Fos reporter probe is determined, for example by SDS-PAGE, andcompared to a cell control containing c-Fos and a normal level of FRKkinase activity.

The Fos protein used in the method of detection of the FRK kinasedescribed above may exist as a single protein unit or a fusion protein.The fusion protein preferably consists of c-Fos andglutathione-S-transferase (GST) as a carrier protein. The c-fosnucleotide sequence is cloned 3' to the carrier protein in an expressionvector, such as pGEX or such derivatives as pGEX2T or pGEX3X, the geneis expressed, the cells are lysed, and the extract is poured over acolumn containing a resin or mixed directly with a resin to which thecarrier protein binds. When GST is the carrier, a glutathione (GSH)resin is used. When maltose-binding protein (MBP) is the carrier, anamylose resin is used. Other carrier proteins and the appropriatebinding resin will be known to those of skill in the art.

The materials of the invention are ideally suited for the preparation ofa kit. The kit is useful for the detection of the level of a c-Foskinase comprising an antibody which binds a c-Fos kinase or a nucleicacid probe which hybridizes to FRK nucleotide, the kit comprising acarrier means being compartmentalized to receive in close confinementtherein one or more containers such as vials, tubes, and the like, eachof the container means comprising one of the separate elements to beused in the assay. For example, one of the container means may comprisea monoclonal antibody of the invention which is, or can be, detectablylabelled. The kit may also have containers containing buffer(s) and/or acontainer comprising a reporter-means (for example, a biotin-bindingprotein, such as avidin or streptavidin) bound to a reporter molecule(for example, an enzymatic or fluorescent label).

The following examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXAMPLES

Ras proteins exert their mitogenic and oncogenic effects throughactivation of downstream protein kinases (Egan, et al., Nature,365:781-783, 1993). A major question is how Ras-generated signals reachthe nucleus to activate downstream target genes. AP-1, a heterodimericcomplex of Jun and Fos proteins, which activates mitogen-inducible genes(Angel, et al., Biochem. Biophys. Acta., 1072:129-157, 1991), is a majornuclear target of Ras (Herrlich, et al., Trends Genet., 5:112-116,1989). Recently, Ras- and UV-responsive protein kinases thatphosphorylate c-Jun on serines (Ser) 63 and 73 and stimulate itstranscriptional activity were identified (Hibi, et al., Genes & Dev.,7:2135-2148, 1993). These proline-directed kinases, termed JNK, arenovel MAP kinases (Derijard, et al., Cell, 75:1025-1037, 1994). A shortsequence surrounding the major JNK phosphorylation site of c-Jun (Ser73)is conserved in c-Fos and is part of its activation domain (Sutherland,et al., Genes & Dev., 6:1810-1819, 1992). The following Examplesdemonstrate that Ras does indeed augment c-Fos transcriptional activitythrough phosphorylation at Thr232, the homolog of Ser73 of c-Jun.However, this is mediated by a novel Ras- and mitogen-responsiveproline-directed protein kinase that is different from JNKs and ERKs.Therefore, at least three types of proline-directed kinases (Thomas,Cell, 68:3, 1992) transmit Ras and mitogen generated signals to thetranscriptional machinery.

Example 1 Regulation of c-Fos Transcriptional Activity

The sequence surrounding the major JNK phosphorylation site in c-Jun isconserved in c-Fos (Sutherland, et al., supra), such that Thr232 ishomologous to Ser73 in c-Jun (FIG. 1A). Both sequences correspond to theHOB1 regions of the Jun and Fos activation domains. To test whether thec-Fos activation domain is Ras-responsive, a fusion was generatedbetween amino acids (AA) 210-313 of c-Fos (containing HOB1 and HOB2) andthe DNA binding domain of GHF-1, a pituitary specific activator (Bodner,et al., Cell, 55:505-518, 1988). FIGS. 1A and 1B show diagramsillustrating the salient features of c-Fos and the GHF1-cFos (210-313)chimera, including the position of the HOB1 and HOB2 regions and asequence alignment of the HOB1 regions of c-Jun and c-Fos.

F9 cells were transfected with 0.85 μg of a -237 rGH-LUC reporter, 0.35μg of pSG-(GHF1-cFos(210-313) or pSG-GHF1-cFos(210-313;S232A), and 0.75μg pSG-Ha-Ras(Leu61) expression vectors per 35 mm well. To constructGHF1-cFos(:210-313), a 1.4 kb HindIII-Hpal fragment from cJ/GHF-1(Binetruy, et al, Nature, 351:122-127, 1991) was cloned into pSG6between the HindIII and Small sites. pSG6 is derived from pSG5 (Green,et al., Nucleic Acids Res. 16:369, 1988) and contains a sequence fromthe HincII to the BamHI site of pBluescript cloned between the EcoRi andBamHI sites of pSG5. The c-Jun coding region was replaced by the Flagepitope (Ellis, et al., Cell, 45:721-732, 1986). The last two codons andthe stop codon of GHF-1 were modified by site-cdirected mutagenesis tocreate an Ncol site. The codons encoding AA209/210 and 313/314 of mousec-Fos were modified by site-directed mutagenesis to generate Ncol andBamHI sites, respectively. The resultant 312 bp NcoI-BamHI fragmentcontaining AA210-313 of mouse c-Fos was inserted between NcoI sitecreated at the end of the GHF-1 coding region and a downstream BamHIsite to generate GHF1-cFos(210-313). GHF1-cFos(210-313;T232A) isidentical to GHF1-cFos(210-313) except that it contains a threonine(Thr) to alanine (Ala) substitution at position 232 introduced bysite-directed mutagenesis. The -237 rGH-LUC reporter was made byinserting a 255bp HindIII-BstYI fragment of rat GH-CAT (West, et al.,Mol. Cell. Biol., 7:119:3-1197, 1987) between the HindIII and BamHIsites in the polylinker region of p20LUC (Felgner, et al., Proc. Natl.Acad. Sci. USA, 84:7413-7418, 1987).

A portion of the cells were treated with TPA (100 ng/ml) or UV (80J/m²), as indicated. The treated cells were harvested 8 hours later andassayed for LUC activity. One unit of relative activity is equivalent tofive-fold activation by GHF1-cFos(210-313) over the basal level ofreporter activity in the presence of "empty" expression vector. Theaverages of four different experiments were determined.

Cotransfection experiments indicated that GHF1-cFos(210-313) activatedthe rGH-LUC reporter and its activity was stimulated 7-fold by oncogenicHa-Ras (FIG. 1C). Both the basal and the Ras-induced activities ofGHF1-cFos(210-313) are similar to those of a cJun-GHF1 chimera(Binetruy, et al., supra). Substitution of Thr232 by an Ala residue hadno effect on the basal activity of GHF1-cFos(210-313;T232A) butabolished its response to Ha-Ras. Since Ha-Ras does not affect GHF-1 DNAbinding activity (Smeal, et al., Mol. Cell. Biol., 12:3507-3513, 1992),the enhancement of GHF1-cFos(210-313) activity is due to potentiation ofthe c-Fos activation function.

In addition to Ha-Ras, the transcriptional activity of c-Jun isstimulated by UV irradiation (Devary, et al., Cell, 71:1081-1091, 1992),which results in strong activation of JNK (Hibi, et al., supra;Derijard, et al., supra). However, activation by GHF1-cFos(210-313) wasnot potentiated by UV irradiation, or 12-0-tetradecanoyl phorbol13-acetate (TPA, FIG. 1C). These results suggested that, despite thesimilarity between the HOS1 regions of c-Jun and c-Fos, the kinase thatphosphorylates Thr232 was different from the JNKs, whose activity isstimulated more efficiently by UV irradiation than by Ha-Ras expression.Since TPA is an efficient activator of ERK1 and 2 (Thomas, et al., Cell,68:1031-1040, 1992), these kinases are also unlikely to be responsiblefor Thr232 phosphorylation.

Example 2 c-Fos Phosphorylation Contributes to AP-1 Activation

Ha-Ras potentiates activation by c-Jun homodimers (Binetruy, et al.,supra; Smeal, et al., supra) and increases endogenous AP-1 activity(Herrlich, et al, supra; Wasylyk, et al., EMBO J., 7:2475-2483, 1988;Schonthal, et al., Cell, 54:325-334, 1988). By dimerizing with c-Jun,c-Fos is an important contributor to AP-1 activity (Chiu, et al., Cell,54541-552, 1988; Sassone-Corsi, et al., Cell, 54:553-560, 1988;Kouzarides, et al., Nature, 336:646-656, 1988; Smeal, et al., Genes &Dev., 3:2091-2100, 1989). Therefore, a c-Jun:c-Fos heterodimer wasexamined to determine whether it also responds to Ha-Ras.

F9 cells were transfected with 0.3 μg of -73 Col-LUC (Deng, et al.,Genes & Dev., 7:479, 1993) and 0.15 μg of either RSV-cJun,RSV-cJun(S63A;S73A), pSV40-cFos (mouse) or pSV40-cFos(T232A) with orwithout 0.3 μg of pSG-Ha-Ras(Leu61) per 35 mm well, as indicated. Theaverages of two experiments (each in duplicate) are shown in FIG. 2.Fold activation was determined by dividing the actual luciferaseactivity obtained in the presence of the Jun and Fos expression vectorsto the activity obtained when the reporter was contransfected with an"empty" expression vector (pRSV-O).

Transactivation of the AP-1 responsive -73Col-LUC reporter by c-Fos plusc-Jun was, indeed, stimulated by Ha-Ras (FIG. 2). Transactivation bywild type (wt) c-Fos in combination with c-Jun lacking the JNKphosphorylation sites, c-Jun(S63A;S73A), and by c-Fos(T232A) plus wtc-Jun was also stimulated by Ha-Ras. However, transactivation of-73Col-LUC by c-Fos(1-232A) plus c-Jun(S63A;S73A) was no longer Ha-Rasresponsive. This result suggests that phosphorylation at Thr232 isrequired for Ha-Ras responsiveness when c-Fos is complexed with itsphysiological partner, c-Jun. Substitution of Thr232 by Ala and/orHa-Ras expression had no effect on accumulation of c-Fos or itsinteraction with c-Jun.

Example 3 Phosphorylation of Thr232 is Ras-Responsive in Vivo

Transient coexpression of oncogenic Ha-Ras with c-Fos resulted inincreased phosphorylation of wt c-Fos but not of c-Fos(T232A).Two-dimensional mapping (Boyle, et al., Meth. Enzym., 201:110-149, 1991)of tryptic peptides derived from wt c-Fos and c-Fos(T232A) confirmedthese observations and indicated that Thr232 is the major Ha-Rasresponsive phosphoacceptor of c-Fos.

F9 cells were transfected with 10 μg of expression vectors encoding wtc-Fos or c-Fos(T232A) in the absence or presence of 10 μg ofpSG-Ha-Ras(Leu61), as indicated. Equal numbers of cells were labelled 12hours after transfection for 4 hours with ³² -p orthophosphate (0.5mCi/ml). c-Fos proteins were purified by immunoprecipitation using amonoclonal antibody (de Togni, et al., Mol. Cell. Biol., 8:2251-2256,1988) and subjected to two-dimensional phosphopeptide analysis, asdescribed (Boyle, et al., supra). Maps were exposed to preflashed x-rayfilms for three days. 1, 2, 3 and 4 refer to major phosphopeptides thatmigrate similarly to phosphopeptides previously shown to be derived fromthe C-terminal region of c-Fos (Tratner, et al., Mol. Cell. Biol,12:998-1006, 1991). R is the phosphopeptide which is most dramaticallystimulated by Ha-Ras (FIG. 3A).

Most of the Ha-Ras mediated increase in c-Fos phosphorylation waslocalized to tryptic peptide R. Phosphopeptide R was missing inc-Fos(T232A), indicating that it arises from phosphorylation of Thr232.Furthermore, the migration position of this peptide is entirelyconsistent with its predicted amino acid composition (Boyle, et al.,supra). The predicted R phosphopeptide is 41 AA long because of cleavagebetween Asp246 and Pro247 by performing acid (Landon, Meth. Enzym.,47:145-149, 1977). This peptide is neutral in the pH1.9 electrophoresisbuffer and has a high R_(f) value (0.46) in the chromatography systemused (Boyle, et al., supra). Furthermore, phosphoaminoacid analysisrevealed the presence of phosphothreonine in the R peptide of wt c-Fos.

To confirm this assignment, the response of a c-Fos(T232S) mutant toHa-Ras was examined. F9 cells were transfected with 10 μg of expressionvectors encoding wt c-Fos or c-Fos(T232S) in the absence or presence of10 μg of pSG-Ha-Ras(Leu61), as indicated. The cells were labeled andprocessed as described above. R and R' refer to the phosphopeptideswhich are most dramatically elevated in Ha-Ras expressing cells and arederived from wt c-Fos and c-Fos(T232S), respectively. Coexpression ofHa-Ras stimulated the phosphorylation of c-Fos(T232S) resulting inappearance of phosphopeptide R' (FIG. 3B).

The R and R' phosphopeptides from the chromatograms shown in FIGS. 3Aand 3B were extracted from the plates and subjected to partial acidhydrolysis and analyzed by two-dimensional high-voltage electrophoresisas described (Boyle, et al., supra). The directions of theelectrophoretic separation are indicated (FIG. 3C). ³² P-labeledphosphoaminoacids were detected by exposure to preflashed x-ray film,using an intensifying screen for 8 days at -80° C. Nonradioactivephosphoaminoacid standards were detected by ninhydrin staining (FIG.3C). Phosphoaminoacid analysis indicated that whereas phosphopeptide Rcontained phosphothreonine, phosphopeptide R' contained phosphoserine(FIG. 3C). Ha-Ras expression also led to a slight and variable increasein phosphopeptides r1 and r2, derived from wt c-Fos, and decreased thelevel of others (FIGS. 3A and 3B). These changes were also observed withc-Fos(T232A). Phosphoaminoacid analysis revealed the presence ofphosphoserine in both r1 and r2.

Example 4 c-Fos is Phosphorylated by a Novel Ras and Mitogen ActivatedKinase

Agents that activate either JNK or ERK1/2, other than Ha-Ras, do notstimulate GHF1-cFos activity as shown in FIG. 1C. To further investigatethis point, the effect of UV and TPA on c-Fos phosphorylation wasexamined. Human c-Jun was prepared as described (Deng, et at., supra)and mouse c-Fos (both wt and mutant) was prepared using the sameexpression system. Both proteins were purified to near homogeneity. Therecombinant c-Jun and c-Fos proteins were incubated at 30° C. for 30minutes with the relevant kinases in kinase buffer (20 mM Hepes pH 7.6,10 mM MgCl₂, 20 mM β-glycerophosphate, 20 mM p-nitrophenylphosphate, 0.1mM NaVO₄, 2 mM DTT) containing 10 μM γ³² P-ATP in a 20 μl volume. Thereactions were terminated by addition of 6.5 μl 4X SDS-PAGE samplebuffer and boiling. Purified ERK1/2 is a mixture of both enzymes (a giftfrom Dr. M. Cobb). Polyclonal anti-JNK1 antiserum was prepared againstrecombinant JNK1 and was used to immunopurify activated JNK1 fromUV-irradiated HeLa cells. Immunoprecipitations were performed asdescribed (Derijard, et al., Cell, 75:1025-1037, 1994).

FIG. 4A shows the analysis of in vivo phosphorylation of c-Fos. Wt c-Fosexpression vector was transiently transfected into F9 cells that weretreated as indicated. C:control; T:TPA (100 ng/ml) treatment for 15minutes before harvest; U:UV irradiation (80 J/m²), 30 minutes beforeharvest; R:cotransfection with pSG-Ha-Ras(Leu61) as described in EXAMPLE3 (FIG. 3). Cells were labeled and processed as described in EXAMPLE 3(FIG. 3). The c-Fos immunoprecipitates were separated by SDS-PAGE andautoradiographed.

By contrast to Ha-Ras, UV and TPA had a marginal effect on c-Fosphosphorylation (FIG. 4A). The small increase in total c-Fosphosphorylation after TPA or UV exposure was restricted tophosphopeptides 2 and 4, and no changes in phosphopeptide R weredetected. TPA, however, activated ERK2, whereas UV irradiation activatedJNK1. It is therefore unlikely that either the ERKs or the JNKsphosphorylate c-Fos at Thr232. This assertion is further supported by invitro phosphorylation experiments.

Recombinant c-Fos or c-Fos(T232A) (50 ng each) was mixed withrecombinant c-Jun (40ng) and incubated at 30° C. for 30 minutes withimmunopurified JNK1 or purified ERK1/2 (a mixture of both enzymes) inkinase buffer containing γ-32-ATP. The reactions were terminated byaddition of 6.5 μl 4xSDS-PAGE sample buffer and boiling. W:wt c-Fos,M:c-Fos(T232A). The phosphorylated proteins were separated by SDS-PAGEand visualized by autoradiography. The bands corresponding to c-Jun andc-Fos are indicated. Using c-Jun:c-Fos heterodimers as substrates,ERK1/2 (a mixture of both enzymes) phosphorylated wt c-Fos andc-Fos(T232A) with similar efficiencies (FIG. 4B).

Two-dimensional tryptic peptide maps of wt c-Fos phosphorylated in vitroby ERK1/2 as described above and a 1:1 mixture between c-Fosphosphorylated in vitro by ERK1/2 and c-Fos labeled in vivo and isolatedfrom Ha-Ras cotransfected cells. As revealed by peptide mappingphosphorylation by ERK1/2 gave rise to phosphopeptides 2 and 4, whichare also phosphorylated in vivo (FIG. 4C). The low level of c-Junphosphorylation by ERK1/2 occurs at Ser243, as previously reported(Alvarez, et al., J. Biol. Chem., 266:15297, 1991). JNK1, on the otherhand, phosphorylated c-Jun very efficiently but did not phosphorylatec-Fos.

Example 5 Regulation of c-Fos Transcriptional Activity by EGF

To identify the Ras-responsive protein kinase that phosphorylates Thr232a cell line, A431, in which c-Ha-Ras is efficiently activated byepidermal growth factor (EGF) (Buday, et al., Cell, 73:611-620, 1993)was utilized. First it was determined whether c-Fos transcriptionalactivity was regulated by EGF.

A431 cells on 60 mm dishes were transfected with 1 μg of a -237 rGH-LUCreporter together with 1 μg of SRα expression vectors encoding:GHF1-cFos(210-313), GHF1-cFos(210-313;T232A), GHF1-cFos(210-313;P233G)or GHF1-cFos(210-313;P233V), using the lipofection procedure (Felgner,et al., supra). After 48 hours cells were either treated with EGF (100ng/ml) or left untreated and harvested 8 hours later for determinationof LUC activity. The averages of three different experiments are shown.Using the GHF1-cFos(210-313) construct, 6-fold stimulation by EGF wasobserved (FIG. 5A). This stimulation is likely to be dependent onphosphorylation at Thr232, as GHF1-cFos(210-313;T232A) was notresponsive to EGF. Thr232 is followed by a Pro residue, suggesting it isrecognized by a proline-directed kinase (Thomas, supra). Indeed,substitution of Pro233 by either a Gly or a Val residue abolished theresponses to EGF (FIG. 5A) and Ha-Ras.

A431 cells on 60 mm dishes were transfected with 1 μg of a -237 rGH-LUCreporter together with 1 μg of SRα expression vectors encodingGHF1-cFos(210-313) or GHF1-cFos(210-313;T232A) in the absence orpresence of 3 μg of SRα-Ras(Asn17), as described above. As suggested byits inhibition by coexpression of the dominant negative Ha-Ras (Asn17)mutant (Smeal, et al., Mol. Cell. Biol., 12:3507-3513, 1992; Feig, etal., Mol. Cell. Biol., 8:3235-3243, 1988), the stimulation of c-Fostranscriptional activity by EGF is Ras-dependent (FIG. 5B).

Example 6 Identification of the Mitogen -Activated KinasePhosphorylating c-Fos AT Thr232

Extracts of EGF stimulated A431 cells were examined for the presence ofa protein kinase activity specific for Thr232 using the in-gel kinaseassay (Kameshita, et al., Anal. Biochem., 183:139-143, 1989). Whole cellextracts (100 μg each) of A431 cells treated with EGF (E, 100 ng/ml for15 minutes) or untreated (C) were separated on SDS-polyacrylamide gelscontaining immobilized c-Fos, GST-cFos(210-313), GST-cFos(210-313;T232A)or GST proteins. The gels were subjected to renaturation and in-situphosphorylation as described (Hibi. et al., supra). The 88 kD bandcorresponding to FRK is indicated. Whole cell extracts of EGF-treated(E), EGF+dexamethasone (D+E) treated or untreated (C) PC12 Ha-Ras(Asn17!cells (Szeberenyi, et al., Mol. Cell. Biol., 10:5324-5332, 1990) wereanalyzed by the in-gel kinase assay as described above with immobilizedc-Fos as a substrate. These cells were either untreated or treated withdexamethasone (10⁻⁶ M) for 3 hours, as indicated, and then exposed toEGF (100 ng/ml), as indicated, for 15 minutes prior to harvesting. Themigration position of FRK is indicated.

Incubation of A431 cells with EGF resulted in rapid activation of an88kD protein kinase activity, termed FRK (Fos Regulating Kinase), thatphosphorylated c-Fos or GST-cFos substrates which were incorporated intothe gel (FIG. 6A). The 88 kD FRK has also been detected in EGF-treatedF9, COS, HeLa and PC12 cells (FIG. 6B). In a PC12 derivative containingan inducible Ha-ras(asn17) allele, (Szeberenyi, et al., supra),activation of FRK by EGF was inhibited by induction of Ha-Ras(Asn17),further substantiating the conclusion it is a Ras-dependent kinase (FIG.6B). FRK activity was not detected in the absence of c-Fos or wheneither c-Jun, GST or GST-cFos(T232A) were used as substrates (FIG. 6A).

In-gel kinase assays using extracts of EGF-stimulated A431 cells andeither wt c-Fos or c-Fos(T232A) substrates were performed as describedabove. The 88 kD bands corresponding to FRK-phosphorylated c-Fosproteins were excised and subjected to tryptic phosphopeptide mapping(Boyle, et al., supra). Shown are the maps of wt c-Fos and c-Fos(T232A)phosphorylated by FRK. Phosphopeptide mapping indicated thatphosphorylation by FRK occurred on peptides R, r1 and r2 (FIG. 6C), thesame phosphopeptides detected after Ha-Ras coexpression (FIG. 3C). Usingfull length c-Fos(T232A) as a substrate, the same kinase activity wasdetected but, as indicated by phosphopeptide mapping, phosphorylationoccurred only on peptides r1 and r2 (FIG. 6C). Therefore, theEGF-activated 88 kD FRK is specific for Thr232 and the two secondaryRas-responsive phosphorylation sites. Since substitution of Pro233 byGly prevented phosphorylation of Thr232, FRK is a proline-directedkinase.

These experiments demonstrate that activated Ha-Ras augments c-Fostranscriptional activity by stimulating its phosphorylation on Thr232.Unexpectedly, the protein kinase that phosphorylated c-Fos at Thr232 isneither JNK1 (46 kD in size) nor JNK2 (55 kD in size), whichphosphorylate c-Jun at the Ras-responsive sites, Ser63 and 73 (Hibi, etal., supra; Derijard, et al., supra). This conclusion is based on theinability of UV irradiation, a potent JNK activator (Hibi, et al.,supra; Derijard, et al., supra), to induce c-Fos phosphorylation invivo, and the failure of immunopurified JNK1 to phosphorylate c-Fos invitro. In addition, TPA, a potent activator of ERK1 and 2 (Thomas, etal., supra), does not stimulate Thr232 phosphorylation in vivo andpurified ERK1/2, although being efficient c-Fos kinases, do notphosphorylate it at Thr232. Therefore, while the mitogen-responsivekinase that phosphorylates c-Fos at Thr232 is proline-directed, like theJNKs and ERKs, it is a different enzyme.

Concurrently, the invention shows that EGF-stimulation results in rapidactivation of an 88 kD protein kinase that phosphorylates c-Fos atThr232. In addition to its EGF and Ras responsiveness, like the otherMAP kinases, this protein kinase, termed FRK, is a proline-directedkinase. Preliminary evidence suggests that the two secondary FRKphosphopeptides (r1, r2) contain Ser133 and are due to cleavage atLys139 and Arg140, respectively. Ser133 is also followed by a Pro and islocated within a sequence similar to that surrounding Thr232. Despitethe size similarity to RSK (Blenis, Cancer Cells, 3:445-449, 1991),another mitogen-responsive kinase that phosphorylates c-Fos (Chen, etal., Proc. Natl. Acad. Sci. USA, 90:10952-10956, 1993), FRK is differentenzyme. First, RSK is not proline-directed (Stokoe, et al., Biochem. J.296:843-849, 1993; Kemp, et al., Trend Biochem. Sci. 15:312-316, 1990),while FRK is. In fact, Pro insertion at position P+1 inhibitsphosphorylation by RSK (Stokoe, et al., supra). Second, the RSKconsensus includes basic residues (Stokoe, et al., supra; Kemp, et al.,supra), which are absent from the immediate surrounding of Thr232.Third, RSK phosphorylates c-Fos at Ser362 (Chen, et al., supra), whichcan be deleted without affecting phosphorylation by FRK.

The foregoing is meant to illustrate, but not to limit, the scope of theinvention. Indeed, those of ordinary skill in the art can readilyenvision and produce further embodiments, based on the teachings herein,without undue experimentation.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..11                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GlyLeuProGluAlaSerThrProGluSerGlu                                             1510                                                                          __________________________________________________________________________

We claim:
 1. An isolated polypeptide:a) having a molecular weight ofabout 88 kD as determined by reducing SDS-PAGE; b) having serine andthreonine kinase activity; and c) phosphorylating the c-Fos activationdomain at amino acid residue Thr 232.